1. Field of the Invention
The invention relates to a process for preparing O-acetyl-L-serine by means of fermentation.
2. The Prior Art
Fermentative processes for preparing amino acids are nowadays commonplace. These processes are, in particular, methods for preparing representatives of the twenty proteinogenic amino acids which are highly relevant from the economic point of view, such as L-glutamic acid, L-lysine and L-threonine. However, there is an increasing number of reports of processes for preparing proteinogenic amino acids which command smaller markets in the range of from 1000 to 10,000 tons per year, such as L-phenylalamine and L-cysteine.
By contrast, scarcely any corresponding methods are known for preparing biosynthetic precursors of the twenty proteinogenic amino acids. However, it is precisely these precursors which can represent interesting products since they frequently possess chiral centers and can serve as building blocks for synthesizing pharmaceutical active compounds. For example, patent application WO 00/44923 describes the preparation of shikimic acid, which is an intermediate in the biosynthesis of aromatic amino acids. Another example is that of application EP 0994190 A2, which reports the use of fermentation to produce L-homoserine, which is a precursor of L-methionine.
O-Acetyl-L-serine is a biosynthetic precursor of L-cysteine. It is an amino acid in its own right and is formed in bacterial and plant metabolism by L-serine being acetylated at the hydroxyl function. This reaction is catalyzed by the enzyme serine O-acetyltransferase [EC 2.3.1.30], which is encoded by the cysE gene. In the cell, O-acetylserine is subjected to further reaction to form L-cysteine. In this reaction, the acetate function is replaced with a thiol function.
Difficulties with preparing o-acetyl-L-serine by fermentation result from the fact that the substance is very labile and isomerizes to N-acetyl-L-serine at pH values above 4.0. At a pH of 7.6, the rate of the reaction is 1%xc3x97minxe2x88x921 (Tai et al., 1995, Biochemistry 34: 12311-12322). This means that no significant quantities of O-acetylserine can be detected under these conditions after a fermentation process which is usually conducted for at least one-and-a-half days. The isomerization reaction is irreversible and its rate increases still further as the pH increases. Because its amino function is blocked, N-acetyl-L-serine can no longer be used for peptide syntheses, in contrast to O-acetyl-L-serine.
Another difficulty is that the level of O-acetyl-L-serine in the cell is very low and is subject to powerful regulation. On the one hand, serine acetyltransferase is inhibited allosterically by L-cysteine, and no synthesis of O-acetyl-L-serine is consequently possible in the presence of xcexcM concentrations of L-cysteine. On the other hand, the isomerization product N-acetyl-L-serine acts as an inducer of the sulfur regulon and thereby leads to the rapid reaction of O-acetyl-L-serine with sulfide to form L-cysteine.
Dassler et al. (2000, Mol. Microbiol. 36: 1101-1112) have reported that cells which overproduce the membrane protein YdeD (=Orf299) secrete L-cysteine, 2-methylthiazolidine-2,4-dicarboxylic acid and also O-acetyl-L-serine into the culture medium. However, it was possible to detect the O-acetyl-L-serine only in very small quantities, of 0.12 g/l, and then only in shaking flask experiments. On the other hand, only N-acetyl-L-serine was obtained in fermentation experiments which enable higher yields to be obtained as a result of improving the nutrient supply. The possibility has been discussed that O-acetyl-L-serine is sufficiently stable only at pH values of 4-5. However, this pH range is not suitable for a fermentative preparation due to the poor growth of neutrophilic bacteria such as Escherichia coli. 
The fermentative preparation of N-acetyl-L-serine at pH 7.0 using Orf299 has also been described in patent application EP 0885962 Al. In this case, the orf299 gene (designated by SEQ.ID.NO:3 in the application) was combined with suitable cysE alleles. These alleles encoded serine acetyltransferases which were subject to less feedback inhibition by L-cysteine. This resulted in an increased production of O-acetyl-L-serine being achieved in the cell and ultimately in the accumulation of N-acetyl-L-serine due to the rapid isomerization.
Using cysE alleles which are subject to less feedback inhibition on their own, as described in application WO 97/15673, does not lead to the accumulation of O-acetyl-L-serine, either. While an increased formation of O-acetyl-serine is achieved intracellularly when this approach is used, only L-cysteine was detected extracellularly and was consequently within reach as a product.
A further serious difficulty when producing O-acetyl-L-serine is the fact, reported by Dassler et al. (2000, Mol. Microbiol. 36: 1101-1112), that overproduction of Orf299 leads to severe impairment of bacterial growth. This is due to the absence of induction of the sulfur regulon because of the intracellular deficiency of the inducer N-acetyl-L-serine.
It is an object of the invention to provide a fermentative process which provides high yields of O-acetyl-L-serine despite the instability of O-acetyl-L-serine and the negative physiological consequences of efficiently exporting O-acetyl-L-serine from the cell.
This object is achieved by culturing, in a fermentation medium, a microorganism strain which is derived from a wild type and in which the endogenous formation of O-acetyl-L-serine and the efflux of O-acetyl-L-serine are increased as compared with the wild type, wherein the pH in the fermentation medium is adjusted to be within the range from 5.1 to 6.5.
It has unexpectedly and surprisingly been found:
that a microorganism strain which is characterized as described above secretes O-acetyl-L-serine in large quantities,
that O-acetyl-L-serine is sufficiently stable in the fermentation medium at pH values of from 5.1 to 6.5, and
that, at the same time, the abovementioned physiological problems of an o-acetyl-L-serine-secreting cell can to a large extent be remedied by supplying O-acetyl-L-serine in increased amounts using feedback-resistant cysE alleles.
The pH of the fermentation medium during fermentation is preferably in the pH range from 5.5 to 6.5; and a pH of from 5.5 to 6.0 is particularly preferred.
Microorganism strains which can be used in the process according to the invention are distinguished by the fact that they
exhibit an increased endogenous formation of O-acetyl-L-serine, and
exhibit an increased efflux of O-acetyl-L-serine.
Strains of this nature are known in the state of the art. An increased endogenous formation of O-acetyl-L-serine can be achieved by introducing modified cysE alleles, as described, for example, in
WO 97/15673 (hereby incorporated by reference) or
Nakamori S. et al., 1998, Appl. Env. Microbiol. 64: 1607-1611 (hereby incorporated by reference) or
Takagi H. et al., 1999, FEBS Lett. 452: 323-327 into a microorganism strain.
These cysE alleles encode serine O-acetyltransferases which are subject to a diminished feedback inhibition by cysteine. As a result, the formation of O-acetyl-L-serine is to a large extent uncoupled from the cysteine level in the cell.
An increased O-acetyl-L-serine efflux can be achieved by increasing the expression of an efflux gene whose gene product brings about the export of O-acetyl-L-serine.
The ydeD gene, which has been described by Dassler et al. (2000, Mol. Microbiol. 36: 1101-1112) and in EP 0885962 A1 (corresponds to the US application with the Ser. No. 09/097,759 (hereby incorporated by reference)) is a particularly preferred efflux gene of this nature.
The modified cysE alleles and/or the efflux gene may be present in the strain employed in single copies or else in increased copy number. They may be encoded chromosomally or be located on self-replicating elements, such as plasmids.
The expression of the genes can be increased, for example, by using suitable promoter systems which are known to a person skilled in the art.
In a preferred embodiment of the present invention, use is made of a microorganism which harbors a cysE allele and/or a ydeD gene, having a native promoter or the gapDH promoter, on a plasmid having a medium-range copy number. An example of such a construct is pACYC184-cysEX-GAPDH-ORF306, which is described in detail in EP 0885962 A1.
In principle, all microorganism strains which are accessible to genetic methods and which can be readily cultured in a fermentation process are suitable for preparing strains of this nature. Preference is given to using bacteria of the Enterobacteriaceae family. Particular preference is given to using organisms of the species Escherichia coli. The preparation of these strains is described in the abovementioned documents and is not part of the present invention.
Strains which are suitable for the process according to the invention are also suitablexe2x80x94as described in EP 0885962 A1 (corresponds to the U.S. Patent application having the Ser. No. 09/097,759 (herewith incorporated by reference))xe2x80x94for preparing cysteine and cysteine derivatives. However, in this case, an adequate supply of an inorganic sulfur source, such as sulfate or thiosulfate, is required in order to obtain optimum quantities of L-cysteine or one of its derivatives.
However, in the process according to the invention, no sulfur source is metered in during the fermentation since the further conversion of O-acetyl-L-serine into cysteine is unwanted. It is only necessary to ensure that an adequate quantity of a sulfur or S source (e.g. sulfate or thiosulfate) is present in the medium so as to cover the requirement of the cellular protein synthesis for cysteine. The adequate quantity in the nutrient medium is preferably from 5 to 50 mM sulfur.
The process according to the invention for preparing O-acetyl-L-serine using a microorganism strain is performed in a fermenter in a manner which is known per se but while setting unusually low pH values during the fermentation.
The microorganism strain is grown in the fermenter as a continuous culture, as a batch culture or, preferably, as a fed-batch culture. Particularly preferably, a carbon or C source is metered in continuously during the fermentation.
Preference is given to using sugar, sugar alcohols or organic acids as the C source. The C sources which are particularly preferably used in the process according to the invention are glucose, lactose or glycerol.
The C source is preferably metered in, in a form which ensures that the content in the fermenter during the fermentation is maintained in a range of 0.1-50 g/l. A range of 0.5-10 g/l is particularly preferred.
The nitrogen or N source which is preferably used in the process according to the invention is ammonia, ammonium salts or protein hydrolyzates. When ammonia is used as the correcting agent for maintaining a constant pH, this N source is then regularly fed in during the fermentation.
Other medium additives which may be added are salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium and iron. Trace amounts (i.e. in xcexcM concentrations), of salts of the elements molybdenum, boron, cobalt, manganese, zinc and nickel, may also be added.
It is furthermore possible to add organic acids, or salts of these acids, (e.g. acetate or citrate), amino acids (e.g. isoleucine) and vitamins (e.g. B1, B6) to the medium.
Examples of complex nutrient sources which may be used are yeast extract, corn steep liquor, soybean meal and malt extract.
The incubation temperature is 15-45xc2x0 C. A temperature of between 30 and 37xc2x0 C. is preferred.
The fermentation is preferably carried out under aerobic growth conditions. Oxygen is fed into the fermenter using compressed air or pure oxygen.
Microorganisms which are fermented in accordance with the process of the present invention which has been described, secrete O-acetyl-L-serine into the culture medium with a high degree of efficiency over a fermentation period of from 1 to 3 days.